Medicaments comprising glicentin as active ingredient

ABSTRACT

Agents for stimulating insulin secretion and for treating diabetes which comprise glicentin as an active ingredient.

FIELD OF THE INVENTION

This invention relates to agents for stimulating insulin secretion andtherapeutic agents for diabetes, which comprise glicentin as an activeingredient and also the invention relates to the use of glicentin astherapeutic agents for diabetes by administration of glicentin tostimulate insulin secretion, thus increasing the blood level of insulin.

BACKGROUND OF THE INVENTION

Glicentin is a peptide comprising 69 amino acid residues, regardless oforigin, as a major component of gut glucagon-like immunoreactants whichare also called gut glucagon-like immunoreactivities (gut GLIs). Thepeptide contains the entire sequence of glucagon in positions 33-61which is extended at the amino terminus via Lys-Arg with the 1-30sequence, glucagon-related pancreatic peptide (or glicentin-relatedpancreatic peptide) and at the carboxy terminus via Lys-Arg with ahexapeptide (positions 64-69) (Volume 11 Gastrointestinal Hormonesedited by V. Mutt, Noboru Yanaihara and Chizuko Yanaihara, pp. 141-162,Academic Press, Inc. 1988).

L. Thim & A. J. Moody have established the primary structure of porcineglicentin (Regulatory Peptides, 2 (1981), 139-150). S. Seino et al havesuggested the amino acid sequences of human, bovine, hamster, rat andguinea pig glicentin from their preproglucagon sequences (FEBS, Vol.203, No. 1, pp. 25-29, 1986). Glicentin and glucagon are produced bytissue specific processing from the same precursor, preproglucagon.Glucagon is formed in pancreas and glicentin in intestine. It is knownthat glucagon counter-acts the blood glucose-lowering action of insulinby stimulating glyconeogenesis and glycogenolysis (R. Ebert et al.,Diabetes Metabolism Review, Vol. 3, No. 1, 1-26 (1987)). Glucagon-likepeptide-1 (GLP-1) (1-37) produced by tissue specific processing from thesame preproglucagon is a 37-amino acid polypeptide hormone and thepeptide derived from it, GLP-1 (7-36 amide) is a 30 amino acidpolypeptide hormone. Those hormones are known to have an insulinreleasing function (T. Matsuyama et al., Diabetes Res. and ClinicalPractice, Vol. 5, 281-284 (1988), D. A. D'Alessio et al., Diabetes, Vol.38, 1534-1538 (1989)).

Some investigators reported that a fraction of Peak II (rich in glucagonof low molecular weight) prepared by fractionation of glucagon-likeimmunoreactivity (GLI) extracted from the mucosa of small intestine indogs exhibited the stimulation of insulin secretion, whereas a fractionof Peak I (rich in glicentin) did not exhibit it (A. Ohneda et al.,Horm. Metab. Res. Vol. 8, 170-174 (1976)).

On the other hand, the biological action of glicentin has not beenconfirmed. It is unknown on what tissue or cell glicentin acts directly.Glicentin so far isolated and purified is an origin of other mammaliananimals than humans. Human glicentin has not been isolated as a purifiedproduct because of the difficulty in an availability of the materialsfor extraction, i.e., human gut. Thus, the physiological roles of humanglicentin have not been elucidated.

The present inventors were successful in synthesizing DNA correspondingto the amino acid sequence of human glicentin which was deduced byGraeme I. Bell et al. (Nature, Vol. 304, 368-371 (1983)) from thesequence of human preproglucagon gene, preparing a recombinant DNAvector using the synthesized DNA and then producing human glicentin froma host cell transformed by the recombinant DNA (Japanese Patent KokaiHei 4-364199). This success leaded to easy availability of humanglicentin in a large amount and as a purified product.

With a rise in the standard of living, the number of diabetic patientsis yearly increasing. The prevalence for the past 30 years shows a rapidincrease tendency as much as 30 times or more. The morbid state ofdiabetes will be caused by an absolute or relative lack of an insulinfunction which plays a central part in the regulation of blood glucose.The main method for the treatment of diabetes includes an alimentarytherapy and an administration of insulin.

Sulfonylurea is known as a drug for stimulating insulin secretion.However, this drug has the disadvantages in that over- orcontinuous-administration leads to a risk of causing hypoglycemia andenough attention is required to keep a normal blood glucose level. Forthe patients suffering from hepatopathy and nephropathy, a special careis further required in the administration, since the drug or itsmetabolite is accumulated. In addition, an administration of the drug togravida is not recommended because of its placental passage and anadministration to a nursing woman is impossible because of its easymigration to milk. Therefore, there is a desire to develop an insulinsecretomotory agent having high safety and less side effects.

SUMMARY OF THE INVENTION

In view of the above situations, the present inventors have made acontinuing study on agents for stimulating insulin secretion and foundthat human glicentin possesses a strong insulin releasing activity, thusleading to the present invention.

The present invention provides an agent for stimulating insulinsecretion which comprises glicentin as an active ingredient. Theglicentin used in the invention can stimulate a secretion of insulinfrom pancreas with no rise in blood glucose and therefore can be usedfor the treatment and prophylaxis of diabetes. Thus the presentinvention further provides a therapeutic agent for diabetes whichcomprises glicentin as an active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents temporal variations in the levels of blood glucoselevel (BGL), insulin (IRI), glucagon (IRG(G21)) and glucagon-likeimmunoreactivity (IRG(G25)) during the administration of glicentin andglucagon in a test animal.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, an administration of glicentin brings aboutthe stimulation of insulin secretion which leads to the treatment andprophylaxis of diabetes caused by the physical condition of insufficientinsulin secretion.

Glicentin which can be used in the present invention includes anyglicentin of an animal origin such as human, porcine, bovine, hamster,rat and guinea pig, as well as glicentin containing methionine (Met)added to the N-terminus, which are prepared by a genetic engineeringprocedure or a synthetic process. Preferably, human glicentin is used inview of an undesirable allergic reaction or the like produced when beingadministered to humans. More preferably, there is used human glicentin(natural type ) not containing methionine (Met) added to the N-terminus.

Human glicentin (natural type) has the following amino acid sequence(SEQ. ID NO:1):

    __________________________________________________________________________    Arg--Ser--Leu--Gln--Asp--Thr--Glu--Glu--Lys--Ser--Arg--Ser--Phe--Ser--Ala-    -Ser--Gln--                                                                   Ala--Asp--Pro--Leu--Ser--Asp--Pro--Asp--Gln--Met--Asn--Glu--Asp--Lys--Arg-    -His--Ser--                                                                   Gln--Gly--Thr--Phe--Thr--Ser--Asp--Tyr--Ser--Lys--Tyr--Leu--Asp--Ser--Arg-    -Arg--Ala--                                                                   Gln--Asp--Phe--Val--Gln--Trp--Leu--Met--Asn--Thr--Lys--Arg--Asn--Arg--Asn-    -Asn--Ile--                                                                   Ala                                                                           __________________________________________________________________________

Further, human glicentin containing methionine (Met) added to theN-terminus has the following amino acid sequence (SEQ ID NO:2):

    __________________________________________________________________________    Met--Arg--Ser--Leu--Gln--Arg--Thr--Gln--Glu--Lys--Ser--Arg--Ser--Phe--Ser-    -Ala--Ser--                                                                   Gln--Ala--Asp--Pro--Leu--Ser--Asp--Pro--Asp--Gln--Met--Asn--Glu--Asp--Lys-    -Arg--His--                                                                   Ser--Gln--Gly--Thr--Phe--Thr--Ser--Asp--Tyr--Ser--Lys--Tyr--Leu--Asp--Ser-    -Arg--Arg--                                                                   Ala--Gln--Asp--Phe--Val--Gln--Trp--Leu--Met--Asn--Thr--Lys--Arg--Asn--Arg-    -Asn--Asn--                                                                   Ile--Ala                                                                      __________________________________________________________________________

The above human glicentin can be prepared by a genetic engineeringprocedure or a synthetic process from a gene of the DNA sequencecorresponding to the above amino acid sequence. An example of thegenetic engineering procedure is a process of producing a desired humanglicentin which comprises preparing a synthetic gene encoding humanglicentin amino acid sequence of the following DNA sequence (SEQ IDNO:3) which has been suggested by the present inventors in JapanesePatent Kokai Hei 4-364199, introducing the synthetic gene into plasmid,transforming E. coli with the resultant plasmid and culturing thetransformant.

    __________________________________________________________________________    5' CGTTCC CTGCAGGACA CTGAAGAAAA ATCTCGTTCT TTCTCTGCTT CTCAGGCTGA              3' GCAAGG GACGTCCTGT GACTTCTTTT TAGAGCAAGA AAGAGACGAA GAGTCCGACT              CCCACTGTCG GATCCAGACC AGATGAACGA AGACAAACGT CATTCTCAGG GTACTTTCAC             GGGTGACAGC CTAGGTCTGG TCTACTTGCT TCTGTTTGCA GTAAGAGTCC CATGAAAGTG             TTCTGACTAC TCTAAATACC TGGACTCTCG TCGAGCTCAG GACTTCGTTC AGTGGCTGAT             AAGACTGATG AGATTTATGG ACCTGAGAGC AGCTCGAGTC CTGAAGCAAG TCACCGACTA             GAACACTAAA CGTAACCGTA ACAACATCGC C 3'                                         CTTGTGATTT GCATTGGCAT TGTTGTAGCG G 5'                                         __________________________________________________________________________

Other processes of producing the human glicentin include introducinginto plasmid a gene of another DNA sequence corresponding to the aboveamino acid sequence of glicentin, transforming E. coli, Bacillussubtilis, yeast or other microorganism with the resultant plasmid andculturing the transformant or alternatively culturing a human glicentinproductive cell. However, it should be understood that human glicentinused in the invention is not limited to one produced by the specificprocess and any human glicentin can be employed in the invention so faras it has the above amino acid sequence.

Usually, glicentin as the active ingredient can be administered orallyor parenterally in the form of suitable pharmaceutical preparations.Such pharmaceutical preparations can be formulated in a conventionalmanner using one or more pharmaceutically acceptable vehicles, adjuvantsand additives, e.g., binders, diluents, solubilizers, stabilizers,buffers, lubricants, coating agents, antioxidants, sweeteners, flavors,colorants and the like. Suitable preparations include powders, granules,tablets, capsules, injections, syrups, suspensions, emulsions or thelike. If necessary, the active ingredient may be administered incombination with other drugs such as sulfonylurea, biguanide or thelike. It may be in bilayered or multilayered tablet with other drugs.The tablets may also be coated with a conventional coating to form,e.g., sugar-coated, enteric-coated or film-coated tablets.

In the formulation of solid preparations such as tablets and capsules,there may be used suitable additives such as lactose, refined sugar,crystalline cellulose, corn starch, calcium phosphate, sorbitol,carboxymethylcellulose, gum arabic, polyvinylpyrrolidone,hydroxypropylcellulose, glycerin, polyethylene glycol, stearic acid,magnesium stearate and talc. In the formulation of liquid preparationssuch as injections and syrups, suitable additives may be used such assodium chloride, sorbitol, glycerin, olive oil, propylene glycol andethyl alcohol.

For a preferred unit dosage form for oral administration, for instance,the aqueous or oily solutions, suspensions or emulsions may containglicentin in an amount of 0.01 to 10 rag, advantageously 0.1 to 1 mg per5 ml and the tablets, capsules or granules may contain glicentin in anamount of 0.01 to 10 mg, advantageously 0.1 to 1 mg.

From the chemical structure, glicentin is considered to undergo adenaturation by an acid within intestine, a decomposition by digestionand a reduction in activity by such denaturation, when administeredorally to human body. Therefore, it is recommendable to release theactive ingredient, glicentin within intestine using an enteric coating.Thus the active ingredient is preferably coated with a conventionalenteric coating agent in the oral administration. The enteric coatingagents include synthetic polymers such as EUDRAGI®, polyacrylate base(available from Rohm Pharma), semisynthetic polymers such as celluloseacetate phthalate or the like.

A preferable administration of glicentin is parenteral for the reason ofits not undergoing denaturation or decomposition. The parenteraladministration includes subcataneous, intravenous, intramuscular andintraperitoneal injections. Glicentin can be formulated into the aqueousor oily solutions, suspensions or emulsions. Preferably, glicentin isadministered in the form of depot preparations for a prolonged effect ofglicentin over a long period of time.

A dose of the active ingredient can be varied depending on the route ofadministration, the symptoms, age, sex and weight of patients and otherfactors, but suitably can be in the range so as to provide a level of100 pM to 10,000 pM in blood. Usual parenteral dosage for adult humanranges from 0.5 μg/kg to 500 μg/kg. However, lower or higher amount maybe administered within the safety range.

When 10 mg/kg of human glicentin (natural type) is intraperitoneallyadministered to male MALB/c mice (6 weeks age), no change in appearanceis observed.

The invention is further illustrated by the pharmacological testdescribed in the following example. Methods and Materials:

Healthy mongrel dogs weighing 13 to 17 kg were subjected to the testafter an overnight fast (A. Ohneda et al., Hotre. Metab. Res., 9,447-452 (1977)).

The animals were anesthetized with sodium pentobarbital (Nembutal®) andtheir abdomens were opened. A cannula for administering test solutionswas inserted into the superior pancreaticoduodenal artery. A cannula forcollecting blood for use in the determination of hormone was insertedinto the superior pancreaticoduodenal vein. After the operation wascompleted, saline solution containing 0.5% arginine was infused into thepancreatic artery at a constant rate of 2 ml/min through the cannulausing an infusion pump. Twenty minutes after the start of infusion, 100pmol or 400 pmol glicentin solution containing 0.2% bovine albumin insaline solution, and then 400 ml glucagon solution containing 0.2%bovine albumin in saline solution were successively infused into thepancreatic artery for 10 minutes at an interval of 40 min. 4 ml portionsof blood samples for hormone assay were collected at various intervalsinto a glass tube containing 1000 KIU aprotinine and 10 mg EDTA.

Measurements:

The plasma insulin was measured in accordance with a known methodmentioned in C. R. Morgan et al., Diabetes, Vol. 12, No. 2, 115-126(1963).

The plasma glucagon was measured using an antiserum (G 21) specific forthe C-terminal portion of glucagon in accordance with the Ohneda et almethod mentioned in A. Ohneda et al., Diabetes, Vol. 24, No. 9, 311-819(1975).

The plasma glicentin was determined as the total glucagon-likeimmunoreactivity using an antiserum (G 25) which cross-reacts with theglucagon related substances in accordance with the Ohneda et al methodmentioned in A. Ohneda et al., Tohoku J. Exp. Med., 129, 207-217 (1979).

The blood glucose was measured for blood drawn from the femoral arteryby the glucose oxidase method using a test kit for the determination ofblood glucose (available from Wako Pure Chemical Industries, Japan underthe trade name of "Glucose B-Test Wako").

Results:

The results are shown in FIG. 1, in which there are demonstrated theeffects of glicentin and glucagon on blood glucose, plasma insulin andplasma glucagon in the arginine loaded dogs. In FIG. 1, the abscissaaxis indicates time in minutes and the infusion of arginine was startedfrom -20 min. Ao shows the time at which glicentin begins to infuse. Theglicentin solution was infused for 10 minutes from Ao min. and 40minutes later, at Bo min., the glicentin solution was infused again for10 minutes and 40 minutes later, at Co min., the glucagon solution wasinfused in the above manner. In FIG. 1, the ordinate axis indicatesblood glucose (GLUCOSE), insulin level (IRI), glucagon level IRG(G 21))and glucagon-like immunoreactivity (IRG(G 25)).

The test results reveal that the administration of glicentin stimulatesinsulin secretion with no change in blood glucose levels. From the factthat the glucagon level is not increased by the administration ofglicentin, it is found that glicentin undergoes metabolism not toconvert immediately to glucagon. Further, it is found that glicentinpossesses a unique effect, insulin release stimulating effect which isdistinct from that of glucagon induced from a rise in insulin levelobserved by the administration of glucagon.

The following examples-illustrate the formulation of typicalpharmaceutical preparations.

Preparation 1 (Tablets)

0.5 g of glicentin, 2 kg of lactose, 20 g of magnesium stearate and 100g of corn starch were mixed, the mixture was compressed, the compressedmixture was pulverized to granules. The granules were formed in atabletting machine to tablets each containing 5.0 μg of glicentin. Thetablets were coated with cellulose acetate phthalate to formenteric-coated tablets.

Preparation 2 (Syrups)

0.1 g of glicentin, 30 g of refined sugar, 26 g of 70% D-sorbitol, 0.03g of ethyl p-oxybenzoate and 0.015 g of propyl p-oxybenzoate weredissolved in 60 g of hot water. After cooling, 0.15 g of glycerin and asolution of the flavor in ethanol were added. Distilled water was addedto the mixture to make up a total amount of 100 ml.

Preparation 3 (Injections)

1 g of glicentin and 99 g of lactose were mixed and the mixture wasdissolved in 1 liter of distilled water for injection. The solution wasfiltered through a sterile filter (e.g., a 0.22 μm membrane filter), 1ml portions of the filtered solution were dispensed into vial bottlesunder sterile condition and freeze dried to provide the preparations forinjection. The preparations are dissolved in distilled water on use.

Preparation 4 (Capsules)

0.5 g of glicentin, 4 kg of lactose, 1.5 kg of crystalline cellulose,1.5 kg of calcium stearate and 3 kg of talc were mixed thoroughly, themixture was compressed, the compressed mixture was pulverized togranules. The granules were encapsuled into two-piece capsules eachcontaining 10.0 μg of glicentin.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgSerLeuGlnAspThrGluGluLysSerArgSerPheSerAlaSer                              151015                                                                        GlnAlaAspProLeuSerAspProAspGlnMetAsnGluAspLysArg                              202530                                                                        HisSerGlnGlyThrPheThrSerAspTyrSerLysTyrLeuAspSer                              354045                                                                        ArgArgAlaGlnAspPheValGlnTrpLeuMetAsnThrLysArgAsn                              505560                                                                        ArgAsnAsnIleAla                                                               65                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetArgSerLeuGlnAspThrGluGluLysSerArgSerPheSerAla                              151015                                                                        SerGlnAlaAspProLeuSerAspProAspGlnMetAsnGluAspLys                              202530                                                                        ArgHisSerGlnGlyThrPheThrSerAspTyrSerLysTyrLeuAsp                              354045                                                                        SerArgArgAlaGlnAspPheValGlnTrpLeuMetAsnThrLysArg                              505560                                                                        AsnArgAsnAsnIleAla                                                            6570                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 207 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: DNA (synthetic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGTTCCCTGCAGGACACTGAAGAAAAATCTCGTTCTTTCTCTGCTTCTCAGGCTGACCCA60                CTGTCGGATCCAGACCAGATGAACGAAGACAAACGTCATTCTCAGGGTACTTTCACTTCT120               GACTACTCTAAATACCTGGACTCTCGTCGAGCTCAGGACTTCGTTCAGTGGCTGATGAAC180               ACTAAACGTAACCGTAACAACATCGCC207                                                __________________________________________________________________________

What is claimed is:
 1. A method for stimulating insulin secretioncomprising: administering an effective amount of glicentin and apharmaceutically acceptable carrier or excipient to an individual inneed thereof.
 2. The method of claim 1, wherein glicentin is humanglicentin.
 3. The method of claim 2, wherein human glicentin has thefollowing amino acid sequence:

    __________________________________________________________________________    Arg--Ser--Leu--Gln--Asp--Thr--Glu--Glu--Lys--Ser--Arg--Ser--Phe--Ser--Ala-    Ser--Gln--Ala--Asp--Pro--Leu--Ser--Asp--Pro--Asp--Gln--Met--Asn--Glu--Asp-    Lys--Arg--His--Ser--Gln--Gly--Thr--Phe--Thr--Ser--Asp--Tyr--Ser--Lys--Tyr-    Leu--Asp--Ser--Arg--Arg--Ala--Gln--Asp--Phe--Val--Gln--Trp--Leu--Met--Asn-    Thr--Lys--Arg--Asn--Arg--Asn--Asn--Ile--Ala.                                  __________________________________________________________________________


4. The method of claim 2, wherein human glicentin has the followingamino acid sequence:

    __________________________________________________________________________    Met--Arg--Ser--Leu--Gln--Arg--Thr--Gln--Glu--Lys--Ser--Arg--Ser--Phe--Ser-    Ala--Ser--Gln--Ala--Asp--Pro--Leu--Ser--Asp--Pro--Asp--Gln--Met--Asn--Glu-    Asp--Lys--Arg--His--Ser--Gln--Gly--Thr--Phe--Thr--Ser--Asp--Tyr--Ser--Lys-    Tyr--Leu--Asp--Ser--Arg--Arg--Ala--Gln--Asp--Phe--Val--Gln--Trp--Leu--Met-    Asn--Thr--Lys--Arg--Asn--Arg--Asn--Asn--Ile--Ala                              __________________________________________________________________________